Independent and coordinate trafficking of single Drosophila germ plasm mRNAs

Abstract

mRNA localization is a conserved mechanism for spatial control of protein synthesis, with key roles in generating cellular and developmental asymmetry. While different transcripts may be targeted to the same subcellular domain, the extent to which their localization is coordinated is unclear. Using quantitative single molecule imaging, we analyzed the assembly of Drosophila germ plasm mRNA granules inherited by nascent germ cells. We find that the germ cell-destined transcripts nanos, cyclin B, and polar granule component travel within the oocyte as ribonucleoprotein particles containing single mRNA molecules but co-assemble into multi-copy heterogeneous granules selectively at the posterior of the oocyte. The stoichiometry and dynamics of assembly indicate a defined stepwise sequence. Our data suggest that co-packaging of these transcripts ensures their effective segregation to germ cells. In contrast, compartmentalization of the germline determinant oskar mRNA into different granules limits its entry into germ cells. This exclusion is required for proper germline development.

Enzymatic production of single-molecule FISH and RNA capture probes

“…As shown in Figure 3C, there was a high degree of overlap between the two colors in the germline, but hardly any puncta were observed in the follicle cell layer with either of the fluorophores. Similarly, another germline specific mRNA, nanos, was detected almost exclusively in the nurse cells; however, no apparent colocalization was observed between the nos18×-Atto633 and osk17×-Atto565, consistent with previous observations (Little et al 2015) indicating specificity of these probe sets (Fig. 3D).…”

“…3A) to that of a set of 45 chemically labeled, conventional smFISH probes (osk3P (45×)-633, Fig. 3B) previously used in smFISH assays (Little et al 2015). We found that our Atto633 labeled probe set-although it contained only 42 different oligonucleotide species-provided a slightly brighter signal than the reference osk3P(45×)-Atto633 set (Fig.…”

“…Furthermore, we observed similar low aspecific backgrounds FIGURE 3. smFISH carried out using 3 ′ end labeled probe mixtures. (A,B) Fluorescent in situ hybridization of oskar mRNA in mid-oogenetic Drosophila egg chambers using a set of 42 enzymatically produced antisense oligonucleotides (A) or a set of 45 chemically labeled smFISH probes (B [Little et al 2015]). Posterior poles, where oskar mRNA localizes, face toward the right of the panels.…”

“…Next, we assayed the single-molecule codetection rate of the two differently labeled probe sets. Since in the nurse cells oskar mRNA was shown to be present in RNPs containing single and two copies of the mRNA (Little et al 2015), we first fitted the signal intensity distribution of the reference channel with Gaussian functions as described in Supplemental Figure S2 and Little et al (2015). We considered that objects with a fluorescence intensity lower than the µ + 2σ of the first fitted Gaussian function contained a single mRNA molecule ( Fig.…”

“…Such studies have identified heterogeneity at all these levels: between cells , subcellular regions (Medioni et al 2012), and between individual RNPs. While some mRNPs contain only a single type and a single copy of an mRNA molecule, others may undergo dynamic homo-or heterotypic oligomerization, resulting in hundreds of copies of the mRNA within a single RNP particle (Little et al 2015). Although today a wide variety of RNA detection techniques with single-molecule sensitivity exists (Gaspar and Ephrussi 2015), the pioneering techniques of singlemolecule fluorescent in situ hybridization (smFISH) were based on an array of short oligonucleotides carrying a welldefined number of labels, thus reducing variance of the detected signal while maintaining a high signal-to-noise ratio (Femino et al 1998;.…”